1. Field of the Invention
The present invention relates to gender identification of eagles, and in particular relates to gender identification of eagles with probe-based real-time PCR.
2. Description of the Related Art
Monitoring the population sex ratio of eagles is essential to prevent extinction. However, efforts to measure population sex ratios for sexually monomorphic birds, including eagles (Ararajuba 2003;11:65-73), often yield male-biased sex ratios (Proc Biol Sci 2004;271(Suppl 5):S321-3-4, Omis Scand 1987;18:122-8, and Curr Ornithol 1989;6:1-50). Many techniques exist for gender identification of monomorphic birds (Yi Chuan 2005;27:297-301). For instance, the Griffiths procedure, which uses the universal P2/ P8 primers (Mol Ecol 1998;7:1071-5), is a common tool for avian gender identification.
The Griffiths procedure is based on the intron length difference between the chromo-helicase-DNA-binding (CHD)-Z and CHD-W gene amplicons. In general, the gender of birds is identified by the P2/ P8-primed PCR, followed by electrophoresis. The CHD-W gene is unique to females, whereas the CHD-Z gene is found in both sexes (i.e., female, ZW, and male, ZZ). Samples with one band are regarded as males, whereas those with two bands are regarded as females. However, intron lengths between the CHD-Z and CHD-W genes usually vary among species (Mol Ecol 1998;7:1071-5, Auk 1998;115:1074-8, and J Avian Biol 1999;30:116-21.). Additionally, due to the limited length difference of the intron for CHD-Z and CHD-W genes, there is accumulating evidence (J Avian Biol 1999;30:116-21, Mol Cell Probes 2004;18:193-6, J Raptor Res 2005;39:286-95, IBIS 2006;148:167-8 and Curr Sci 2007;92:659-62) that the gender of some avian species cannot be accurately determined by the PCR-based protocol alone. Specifically, the length difference in some eagles is extremely short (approximately 3 to 9-bp). Thus, several solutions have been proposed, such as using re-designed primers of the PCR (J Avian Biol 1999;30:116-21 and BMC Biotechnol 2008;8:12.,13), PCR-restriction fragment of length polymorphism (RFLP) (Mol Cell Probes 2004;18:193-6 and Curr Sci 2007;92:659-62) and random amplified polymorphic DNA (RAPD) fingerprinting (Theriogenology 2007;67:328-33 and Theriogenology 2006;65:1759-68). However, the methods are unable to provide universal primers for high-throughput gender identification for multiple species of eagles.